The sphingolipidoses are a group of genetic disorders of glycolipid metabolism, due to lysosomal enzyme deficiencies, that can result in mental retardation and are often fatal. Current studies of these diseases involves a deeper understanding of their precise genetic basis, including their localization within the genome and their molecular biology. The availability of an enzymatically authentic mouse model (the twitcher mouse) for Krabbe's disease, a galactosylceramide beta-galactosidase deficiency in humans, greatly facilitates basic studies on these disorders and provides an animal system for testing gene and enzyme replacement therapies. The proposed studies use the galactocerebrosidase-deficient twitcher mouse to localize the structural gene for this enzyme in the human genome using somatic cell genetics techniques. Cell lines from skin samples of 3 day neonate twitcher mice have been established and continuously propagated in this laboratory for one and one-half years (over 200 cell doublings). These permanent galactocerebrosidase-deficient lines will be crossed with normal human diploid cells exhibiting galactocerebrosidase activity and made neomycin resistant with a retrovirus carrying the dominant gene for this trait. Hybrid cells will be selected in neomycin (G418) medium in the presence of oubain which will eliminate both parental cell types. The hybrids will then be cloned and individual clones assayed for galactocerebrosidase activity and human isozyme contents. The single human isozyme marker shared by a panel of galactocerebrosidase-positive clones will provide the first evidence for the chromosmal localization of the gene for this enzyme in the human genome, since any galactocerebrosidase activity in these hybrid cells must necessarily have come from the human gene. The proposed strategy is unique and avoids the use of immunological or electrophoretical methods for distinguishing the human from the mouse enzyme in human X mouse hybrids. This considered an extremely desirable feature of the twitcher mouse for use in these studies, since the enzyme galactocerebrosidase has been particularly difficult to purify for reliable antibody production and also, cannot be distinguished specifically using the histochemical methods for electrophoresis studies. Future work with these cells will include regional mapping of the gene using chromosmal banding techniques and gene isolation studies.